: There is much interest in the role of oxidative stress as contributing to mechanisms by which ethanol is hepatotoxic. Induction of CYP2E1 by ethanol appears to be a major pathway by which ethanol produces a state of oxidative stress. Besides ethanol, CYP2E1 oxidizes many other important compounds and is induced under a variety of pathophysiological conditions. A major level of regulation of CYP2E1 is at the posttranscriptional stage as several ligands for CYP2E1, including ethanol protect the enzyme against degradation by uncharacterized intracellular proteolytic pathways. The goal of this application is Specific Aim 1 will evaluate the role of the proteasome complex in CYP2EI degradation in human hepatocytes. Turnover of CYP2E1 in cultured human hepatocytes will be studied. The major proteolytic pathways, and whether ubiquitination is required for CYP2E1 degradation will be determined, as will the effects of ethanol on this process. Specific Aim 2 will evaluate the role of reactive oxygen species in triggering CYP2E1 degradation by the proteasome complex. The ability of a variety of antioxidants to alter CYP2E1 levels and turnover in intact cells and reconstituted systems will be evaluated, and evidence for CYP2E1 oxidative modification will be provided. Specific Aim 3 will evaluate the effect of molecular chaperones on steady state levels and turnover of CYP2E1. Experiments will involve studying the effects of inhibitors of heat shock proteins or antibodies or of irnmunodepletion followed by readdition of the modulator in the reconstituted system, or adding inhibitors or addition of plasmids expressing the putative modulator to intact cell models. Does ethanol and other CYP2E1 ligands block the effect of the modulator? These experiments will provide new information on the role of the proteasome and ubiquitination in CYP2E 1 turnover in human hepatocytes, how ethanol protects CYP2E1 against degradation, and identify factors which may trigger rapid turnover of this enzyme. Modulators of any of these newly identified steps may have therapeutic implications for individuals with high levels of CYP2E1.